Cell migration is essential for many diverse physiological processes from embryonic morphogenesis to adult wound healing, as well as being a primary aberrant behavior in pathological processes including tumor metastasis. Great strides have been made over the past four decades in understanding basic genetic and signaling cues underpinning both normal development and tumorigenesis. Yet the understanding of cell migration in these contexts remains rudimentary in comparison. Actin-based protrusion has long been identified as a critical physical requirement for cell migration over a 2D substrate. The Arp2/3 complex is a well-characterized macromolecular machine that assembles branched actin within these protrusions and is required for cell migration. This proposal aims to better understand normal and aberrant cell migration by identifying the factors responsible for recruiting and stabilizing the Arp2/3 complex, as well as the specific amino acids in the complex that allow it to bind actin. An additional aim, making use of newly generated Arp2/3-deficient cells, is to understand the interplay between distinct actin organization paradigms and their impact on higher order cellular processes, including cell motility in vivo. The objectives outlined above will be assessed using a variety of classic and novel cell biological approaches. Arp2/3 deficient or knockout cells have been rescued via re-expression of wildtype or mutant subunits tagged to the fluorescent protein GFP, and will be used to assay Arp2/3 dynamics via FRAP, leading edge protrusion rates in live cells via kymography, to track random cell motility over time, and to test directional migration induced in response to applied gradients of growth factors or adhered matrix cues. Further analyses will involve direct assessment of the actin cytoskeleton via electron microscopy in WT vs. Arp2/3-deficient cells, or GFP-actin dynamics via live cell imaging and FRAP. Finally, stable shRNA or transient siRNA-mediated depletion of other actin-associated proteins will give insight into actin polymerization in the absence of Arp2/3. This work will identify factors that impact actin branch stability and enhance the understanding of Arp2/3 function and the interplay between Arp and non-Arp actin polymerizing proteins during cell motility. Furthermore, advanced imaging approaches and genetic p34 knockout mice established during these studies will lead to a better understanding of the importance of Arp2/3 and non-Arp actin nucleation in additional contexts, such as tumor cell invasion and metastasis.